KR20080003402A - Stilbazium research assays - Google Patents

Abstract

Methods for determining the presence, absence, health or interaction between cells analytes, nucleic acids or microorganisms in a sample are provided. Compounds that fluoresce and kits containing the same are also provided.

Description

Steelbazium research assays

This application is incorporated by reference in U.S. Provisional Patent Application No. 60 / 669,615, filed Apr. 8, 2005, U.S. Provisional Patent Application No. 60 / 734,518, filed Nov. 8, 2005, and filed on Feb. 13, 2006. Priority claims are made based on US Provisional Patent Application 60 / 773,366. The disclosure of the above applications is hereby incorporated by reference in its entirety.

The present invention relates to compositions, methods, and kits for use in assays. More specifically, the present invention relates to the use of stilbazium compounds and their analogs as fluorescent, staining or tag for use in the assay.

One of the most frequently used molecular biological techniques for detecting homologous nucleic acid sequences is nucleic acid hybridization, ie DNA / DNA, RNA / RNA or RNA / DNA hybridization. In this technique, the nucleic acid (DNA or RNA) used as a probe is labeled and the labeled nucleic acid hybridizes to the nucleic acid (DNA or RNA) to be detected. If a nucleic acid used as a probe has homology to the nucleic acid to be detected, each single-stranded nucleic acid hybridizes to its complementary sequence to form a double-stranded sequence, which double-stranded sequence is attached to a labeled probe. Is detected.

Identify, mark, isolate, or modify analytes, microorganisms, amino acid or nucleic acid sequences (e.g., multiple pathogens or multiple genes or multiple genetic variants) in blood or other biological fluids Increasingly, the need to make decisions, or decisions, is increasing in many medical fields. Most multi-analyte assays, such as assays that detect multiple nucleic acid sequences, include multiple steps and provide low sensitivity, limited dynamic range (typically 2 to 100-fold differences). And / or often use sophisticated devices.

As the human genome has been identified, analyzes may be performed to determine the presence of specific sequences, to distinguish alleles in homozygote and heterozygote, to determine the presence of mutations, to evaluate cell expression patterns, etc. There are many opportunities. In many of these cases, one wants to determine a number of different features for the same sample in a single reaction. In many analyses, it is of interest to determine the presence of a particular sequence, whether it is a genomic sequence, a synthetic sequence, or a cDNA sequence. Such sequences may be specifically associated with genes, regulatory sequences, repeats, multimeric regions, expression patterns, and equivalents thereof. Also of interest is determining the presence of one or more pathogens. The need to identify and quantify multiple bases or sequences, potentially distributed on the centimorgan of DNA, presents a major challenge. Ideally, the method is accurate and limits the amount of reagents required to be reasonably economical and / or to discriminate and quantify multiple genes, and / or to determine single nucleotide polymorphism (SNP), and / or at the RNA or protein level. It provides a multiplex analysis that enables the gene expression of.

Radioactivity is being used as a major read-out in early drug discovery assays. However, the need for more information, higher throughput, and miniaturization has led to the transition to fluorescence detection. Fluorescence-based reagents can yield more robust, multi-parameter analyzes with higher throughput and information content and will require lower doses of reagents and test compounds. Fluorescence is also safer and less expensive than radiation-based methods.

Another technique for detecting biological compounds is fluorescence in-situ hybridization (FISH). Swiger et al. (1996) Environ. Mol. Mutagen. 27: 245-254; Raap (1998) Mut. Res. 400: 287-298; Nath et al. (1997) Biotechnic. Histol. 73: 6-22. FISH enables the detection of certain target oligonucleotides, such as DNA or RNA, in a cell or tissue preparation, for example by microscopic visulaization. Thus, FISH is an important tool in such fields as molecular cytogenetics, pathology and immunology in both clinical and basic field laboratories. FISH involves fluorescent tagging of oligonucleotide probes to detect specific complementary DNA or RNA sequences. Specifically, FISH includes incubating an oligonucleotide probe comprising an oligonucleotide complementary to at least a portion of the target oligonucleotide with a cell or tissue sample that includes or is suspected of containing the target oligonucleotide. Detectable labels, such as fluorescent dye molecules, are bound to oligonucleotide probes. Fluorescence signals generated at the hybridization site are typically visualized using an epi fluorescence microscope. An alternative approach is to visualize hybridization of the probe to DNA targets using oligonucleotide probes conjugated to antigens such as biotin or digoxygenin and fluorescently labeled antibodies to such antigens. Many FISH formats are known in the art. For example, Dewald et al. (1993) Bone Marrow Transplantation 12: 149-154; Ward et al. (1993) Am. J. Hum. Genet. 52: 854-865; Jalal et al. (1998) Mayo Clin. Proc. 73: 132-137; Zahed et al. (1992) Prenat. Diagn. 12: 483-493; Kitadai et al. (1995) Clin. Cancer Res. 1: 1095-1102; Neuhaus et al. (1999) Human Pathol. 30: 81-86; Hack et al., Eds., (1980) Association of Cytogenetic Technologists Cytogenetics Laboratory Manual. (Association of Cytogenetic Technologists, San Francisco, Calif.); Buno et al. (1998) Blood 92: 2315-2321; Patterson et al. (1993) Science 260: 976-979; Patterson et al. (1998) Cytometry 31: 265-274; Borzi et al. (1996) J. Immunol. Meth. 193: 167-176; Wachtel et al. (1998) Prenat. Diagn. 18: 455-463; Bianchi (1998) J. Perinat. Med. 26: 175-185; And Munne (1998) MoI. Hum. Reprod. See 4: 863-870.

Thus, differentiation and / or quantification of single or multiple genes, characterized by higher sensitivity, broad dynamic range, better fluorescence, higher degree of multiplexing and / or less stable reagents Assays for, and / or SNP determination, and / or gene expression at the RNA or protein level may increase the simplicity and / or reliability of multianalyte assays and reduce their cost.

In addition, there is a continuing need for labels with the following characteristics in the analytical art: (i) high fluorescence intensity (for small amounts of detection), (ii) suitable separation between absorption and emission frequencies, (iii) desirable solubility (iv) Gaussian-like luminescence of (iv) the ability to easily link to other molecules, (v) stability to harsh conditions and high temperatures, and (vi) symmetrical to multiple color deconvolutions. Linear (symmetric, nearly Gaussian emission lineshape), and / or (vii) compatibility with automated analysis.

Embodiments of the present invention are assays that determine the presence or absence of cells, analytes, nucleic acids or microorganisms in a sample suspected of containing cells, analytes, nucleic acids or microorganisms, wherein the assays comprise stained cells, analytes, A dye that directly stains the cell, analyte, nucleic acid, or microorganism to provide a stained sample comprising the nucleic acid or microorganism, wherein the dye is a compound represented by Formula I below:

In formula I, the NR ₁ R ₂ and NR ₃ R ₄ moieties are in the ortho, meta or para position; X ⁻ is an anionic salt; R _1, R _2, R _3, or R ₄ is C _{1 -10} alkyl (straight or branched), alkenes (linear or branched) with independently selected from the group consisting of, or taken together R ₁ and R ₂ or R ₃ and R ₄ together with the nitrogen atom to which they are attached form a pyrrolidino or piperidino ring; R ₅ is a polyalkylene glycol moiety, C _{1 -10} alkyl (straight or branched), alkenes (linear or branched), alkynes, substituted aryl moiety and unsubstituted aryl moieties, substituted benzyl Moieties and unsubstituted benzyl moieties and / or organometallic moieties, or R ₅ may also be an organometallic compound such as organotin, organosilicon, or organogermanium, and R ₅ is n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and R ₆ is selected from the group consisting of propyl, butyl, or any alkyl compound (CH ₂ contacting the sample with a labeling reagent, which may be _n- MR ₆ , to form labeled cells, nucleic acids or microorganisms; Contacting the stained sample; And observing accumulation of the stained cells, analytes, nucleic acids or microorganisms. Such assays may include cellular assays, chemical assays and biological assays. The dye may be loaded into lipid vesicles or suitable biomaterials. In particular, the dyes may be loaded into microcapsules, liposomes, micelles and / or bicells to form microencapsulated preparations, liposome preparations, micelle preparations and / or vissel preparations, respectively. In addition, the dye may be pegylated.

Embodiments of the invention may include probes comprising a ligand or antibody and a compound of formula I. Probes can be used to detect cells, analytes, nucleic acids and microorganisms.

Embodiments of the invention also include methods of selecting analytes that bind to a compound of Formula I.

Embodiments of the present invention include a method of determining the presence or absence of one or more target compounds, which are compounds of formula I or fluorescent molecules, in a sample, wherein the steps of the methods are specific for the one or more target compounds and are each target-binding, respectively. Providing a plurality of electrophoretic probes having a moiety; Combining the sample with the plurality of probes to form a complex between each target compound and one or more electrophoretic probes specific thereto in the presence of a target compound; And isolating and identifying said compound to determine the presence or absence of said one or more target compounds.

Embodiments of the present invention also include kits for staining cells, analytes, nucleic acids and / or microorganisms.

details

The foregoing and other aspects of the invention will now be described in more detail through other embodiments described herein. It is to be understood that the invention may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art to which this invention pertains.

The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the embodiments of the invention and in the description of the appended claims, the singular forms “a, an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. . Also, as used herein, “and / or” means and encompasses all possible combinations of one or more related enumerated items. As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include both X and Y. As used herein, a phrase such as "between about X and Y" means "between about X and about Y." As used herein, a phrase such as "between about X and Y" means "about about X and about Y." As used herein, "about X Phrases such as "from about X to Y" mean "form about X to about Y". Unless defined otherwise, all terms including technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

All publications, patent applications, patents, and other references cited herein are hereby incorporated by reference in their entirety for teaching related to the sentences and / or paragraphs in which the references are cited.

"Alkyl" as used herein alone or as part of another functional group means a straight or branched hydrocarbon comprising 1 to 10 carbon atoms. Representative examples of alkyl are methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n -Hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and equivalents thereof, including, but not limited to.

As used herein, "lower alkyl" refers to a straight or branched hydrocarbon functional group containing from 1 to 4 carbon atoms. Representative examples of lower alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, and equivalents thereof.

Alkyl and lower alkyl groups are unsubstituted or one or more halo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy, alkenyl Oxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocycloalkyloxy, mercapto, alkyl-S (O) _m , haloalkyl-S (O ) _m , alkenyl-S (O) _m , alkynyl-S (O) _m , cycloalkyl-S (O) _m , cycloalkylalkyl-S (0) _m , aryl-S (O) _m , arylalkyl -S (O) _m , heterocyclo-S (O) _m , heterocycloalkyl-S (O) _m , amino, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkyl Amino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted-amino, acylamino, And may be substituted with a substituent that is acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyano and m is 0, 1, or 2.

As used herein, alone or as part of another functional group, “alkoxy” means an alkyl group, as defined herein, bound to a parent moleculte moiety through an oxy group. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and equivalents thereof.

As used herein, alone or as part of another functional group, "acyl" or "alkanoyl" refers to -C () wherein R is a suitable substituent such as alkyl, alkenyl, alkynyl, aryl, alkylaryl, etc. O) R means a radical.

As used herein, "cell" refers to the basic components of a living individual or a fixed individual and includes organelles. Thus, detecting the presence of a cell, analyzing the cell, staining the cell, and the like may refer to an entire cell or one or more organelles of the cell. According to an embodiment of the invention, the cell may be a plant cell or an animal cell. As recognized by one of ordinary skill in the art to which this invention pertains, as used herein, "cell organelle" refers to a cellular component or structure suspended in the cytoplasm, including those that provide a boundary and have specialized functions. The organelles are the nucleus, smooth endoplasmic reticulum and / or rough endoplasmic reticulum, centrosome, cytoskeleton, cell wall, cell membrane, flagella, cilia, chloroplast, mitochondria, Golgi, ribosomes, lysosomes, centrioles, Include, but are not limited to, acrosomes, glyoxysomes, secretory vesicles, peroxysomes, vacuoles, melanosomes, myofibrils and parenthesomes It is not limited.

As used herein, "dye" means a material that imparts color and / or fluorescence and / or is quantifiable or distinguishable. Color and / or fluorescence can be temporary, semi-permanent or permanent.

As used herein, "analyte" refers to a substance or chemical component that is the subject of an analysis. For example, an analyte can be a molecule, protein, chemical substance, or the like that can be detected as a result of biological, chemical or clinical testing to assess molecules, proteins, chemicals, and the like. Analytes are ions; Metabolites such as glucose and urea; Trace metabolite such as hormones, drugs, steroid hormones; Gases such as breathing gas, anesthetic gas, toxic gas and flammable gas; Toxic vapors; Proteins and nucleic acids; Antigens and antibodies and microorganisms, but are not limited thereto.

As used herein, “nucleic acid” means oligonucleotides, nucleotides, or polynucleotides, and DNAs or RNAs or chimeras thereof that are single- or double-stranded, and may be fully or partially synthetic or natural. . Nucleic acids can include modified nucleotides or nucleotide analogs. Nucleic acids can also be derived from species of origin, including mammalian species, such as plant species or humans, non-human primates, mice, rats, rabbits, cattle, goats, sheep, horses, pigs, dogs, cats, and the like. In some embodiments, the nucleic acid is an isolated nucleic acid. As used herein, an “isolated” nucleic acid is separated from, or substantially substantially separated from, some component of a natural individual, for example, a cellular structural component or other polypeptide or nucleic acid found to be associated with the nucleic acid. It means a nucleic acid that does not contain.

As used herein, “lipid vesicle” means a cellular structure comprising an amphiphile, eg, a surfactant or a phospholipid, characterized by the presence of an internal void. . The internal cavity may be filled with a suitable material such as lipids, aqueous solutions, gases, gels, solid materials, or mixtures thereof. Lipid vesicles are liposomes, helices, discs, tubes, toruss, hexagonal structures, phase structures, micelles, gel phases, reverse micelles, bicelles, microemulsions, Including but not limited to emulsions and combinations thereof.

As used herein, "microorganism" refers to a microscopic entity that can only exist as a single cell or cell cluster.

Embodiments of the invention include compounds of formula I or solvates thereof:

In the above, X ⁻ is an anionic salt,

_{R 1, R 2, R 3} , or R ₄ are independently selected from the group consisting of methyl, ethyl, C _{1 -10} alkyl (straight or branched), alkenes (linear or branched), or taken together R ₁ and R ₂ or R ₃ and R ₄ together with the nitrogen atom to which they are attached form a pyrrolidino or piperidino ring. X ⁻ is fluoride, chloride, bromide, iodide halide, mesylate, tosylate, naphthylate, nosylate, para-aminobenzoate, benzenesulfonate, besylate, lauryl sulfate, 2,4-dihydroxy benzophenone, 2- (2-hydroxy-5'-methylphenyl) benzotriazole, ethyl 2-cyano-3,3-diphenyl acrylate and 5-butyl phenyl salicylate It may be selected from the group containing. R ₅ is a polyalkylene glycol moiety, C _{1 -10} alkyl (straight or branched), alkenes (linear or branched), substituted and unsubstituted aryl, substituted and unsubstituted benzyl, and / or an organometallic moiety to be. R ₅ may also be an organometallic compound such as organotin, organosilicon, or organomagnesium. Additionally, R ₅ is n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and R ₆ is selected from the group consisting of propyl, butyl, or any alkyl compound (CH ₂ ) H-MR ₆ .

In some embodiments of the invention, the compound has the structure:

In another embodiment of the invention, the compound has the structure:

In another embodiment of the invention, the compound has the structure

Stilvaxyl chloride and other salts, analogs and homologs thereof are present as bright red to dark red or other colored compounds which may include UV-visible chromophores and may exhibit characteristic strong fluorescence. Can be.

Stilbare chloride and its other salts, analogs and homologues exhibit typical staining properties and bind to cloth, paper, wood plastic, glass, metal and other substrates, and skin and related biological tissues while retaining their red or pink or other colors. Can be. Stilvaze chloride and other salts, analogs and homologs thereof are prepared by biopolymers such as single- or double-stranded deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) by covalently binding to nucleophilic groups on DNA and RNA chains. biopolymers).

One mechanism of action may include the formation of a strong covalent bond between the nucleophile (Nu :) and one of the stilbare chloride side chains. The remaining side chains retain the chromophores and fluorophores as indicated in the chart below.

Scheme 1.

Nucleophiles can be nucleic acids, microorganisms, amino acids, cells, or analytes. Further, in certain embodiments, nucleic acids, amino acids, or analytes are added to nucleophiles before nucleophiles, such as OH, O ⁻ , NH ₂ , and equivalents thereof, are covalently bound to stilbare chloride, salts, analogs and homologues thereof. Can be combined.

The compounds of the present invention may exist as geometric isomers. Such compositions, which are individual compounds or mixtures, are included within the scope of the invention for their industrial use. E, E-isomer is a structure (configuration) of the present invention, E, E - the seesaw Id (cisoid) and transfected small beads (transoid) 2,6- conformation (conformation) of the structure, it is possible both. Additionally, in addition to the para and meta structures exemplified above, ortho conformations of these structures can be formed. The ortho conjugation structure may include the same salts and moieties as disclosed above and disclosed throughout the present application.

The compounds of the present invention can act quickly to stain objects of interest within less than 10 minutes, and in some embodiments within seconds. Compounds of the invention are observable under bright light and / or fluorescence. In addition, the compounds of the present invention can provide non-toxic staining options for viable cells and provide repeatable staining of viable cells. In addition, the compounds of the present invention can be used in multiple assays of the same culture. Embodiments of the invention may also provide for the detection of mitosis of living cells. The compounds of the present invention are user safe and user friendly. In addition, the compounds of the present invention may be stable for a time sufficient to allow a suitable assay to be performed at room temperature.

Embodiments of the present invention may also include compounds represented by Formula I encapsulated, formulated and / or PEGylated (PEG) in lipid vesicles such as liposomes, micelles and bicells. As used herein, "encapsulated" means a formulation of a compound according to the invention that is encapsulated by a material or substance. The material or material may be of synthetic or natural origin. Thus, lipid vesicles provide a representative mechanism for encapsulation. In addition, as will be understood by one of ordinary skill in the art to which this invention belongs, the compounds of the present invention may be further encapsulated by the application of a coating surrounding the compound. Such coatings may include biomaterials and further include materials discussed below with respect to microcapsules.

The amount of the compound encapsulated, formulated and / or pegylated in the lipid vesicles can be determined by one skilled in the art to which this invention belongs based on how the compound is used. Generally, the compound may be present in an amount of about 1% to about 50% by weight or more of the formulation. As such, the encapsulated, lipid vesicle formulation and pegylated formulation may be water soluble.

1. Microencapsulation ( microencapsulation )

According to some embodiments, the compounds of the present invention may be encapsulated in microcapsules. As used herein, the term "microcapsule" is intended to consider single molecules, encapsulated discrete particles, multiparticulate, liquid multicore, and homogeneously dissolved active ingredients. Encapsulation methods can provide a water soluble or fat soluble active ingredient encapsulated in a shell matrix of a water soluble or fat soluble material. The microencapsulated active ingredient may be protected from oxidation (eg UV) and hydration and may melt, rupture, biograding or dissolve the surrounding shell matrix, or It can be released by slow diffusion through the matrix. Microcapsules typically fall within the size range of about 1 to 2000 microns, although smaller and larger sizes are known in the art.

The compounds of the present invention may be placed in the microcapsule or hollow fiber type used for dispensing. They may also be dispersed in the polymeric material or kept as a liquid.

Microcapsules may be selected from polyethylene, polypropylene, polyester, polyvinyl chloride, tristarch acetate, polyethylene oxide, polypropylene oxide, polyvinylidene chloride or fluoride, polyvinyl alcohol, polyvinyl acetate, urethane, It can be made from a variety of materials, including polycarbonates, and polylactones. Further details on microencapsulation are disclosed in US Pat. Nos. 5,589,194 and 5,433,953. Microcapsules suitable for use in the base material of the present invention have a diameter of about 1.0 to about 2,000 microns.

There are no specific restrictions placed on the form for holding the active ingredient. In other words, there are various formulations that receive the active ingredient by means of a holding mixture. Specific examples include the surface of the active ingredient being covered by the aqueous mixture; Products processed into the desired form, each kneading the active ingredient in the aqueous mixture or forming a homogeneous solution of the aqueous mixture with the aqueous mixture, or dispersing the active ingredient in the aqueous mixture by removal of a solvent or the like. To; Microcapsules which are obtained by processing the dispersion into a desired form such as single molecule, molecular chain, liquid, sphere, sheet, film, rod, pipe, thread, tape or chip. do. In addition, such treated products having a surface covered with a barrier layer for controlling the release of the active ingredient and products coated with an adhesive to improve applicability can be given by way of example. As a further example, the steps of filling an active ingredient contained in a processed mixture in the form of a capillary tube, heat sealing both ends of the capillary tube and then encapsulating the active ingredient therein Product obtained by; And a product obtained by cutting the capillary tube described above in two pieces at the center, whereby each has an opening at one end.

The container is formed by an aqueous mixture having the active ingredient in liquid phase enclosed therein to ensure uniform release over a long period of time. As such a form, generally a tube-, bottle- or bag-shaped container is used.

When the mixture is molded into a container, the sustained release layer preferably has a thickness of at least about 0.002 mm for stable sustained release. If the continuous emissive layer has a thickness not less than about 0.002 mm, no particular problem arises, but a continuous emissive layer in the range of about 0.005 mm to 5 mm can be used. If it exceeds about 5 mm, the amount of the compound released tends to be too small.

In the case of a solid, the release surface area of the sustained release formulation formed from such a container is preferably at least .001 cm ² . A range of .01 cm ² to 1 cm ² may be used.

If the active ingredient is enclosed and received in a container of a sustained release formulation, and the container is formed from an aqueous mixture, the active ingredient may be enclosed in portions. The entrapped amount may be about 0.5 mg to 5 mg, and may be about lmg, 2 mg, 3 mg, or 4 mg.

In the form of a container formed of an aqueous mixture, tubes, bottles and bags can be used. For tube-shaped preparations, tubes with an inner diameter of about 0.4 mm to 10 mm can be used. Internal diameters less than about 0.4 mm make it difficult to fill the container with active ingredients, whereas internal diameters greater than about 10 mm make encapsulation difficult. Bottle-shaped formulations are formed by blow molding or injection molding and generally have an internal volume of about 0.1 to 200 ml. Bottles with an internal volume of less than about 0.1 ml cannot be easily formed, and bottles with an internal volume of greater than about 200 ml are not economical because of the large difference between the amount of active ingredient filled therein and the internal volume. For bag-type preparations, the amount of active ingredient filled inside the bag is preferably about 1 mg to 100 g.

In some embodiments, the total microcapsule composition may comprise about 40-90% liquid fill and about 10-40% shell wall, wherein the liquid fill is about 5-60% Compound, about 25-50% biological synergist and 20-40% water-immiscible organic solvent, the shell being 0.5-20% as its essential component Photostable ultraviolet light absorbent compounds of which all percentages are based on the weight of the total microcapsule composition.

The compound is kept in microcapsules but the composition is packaged and stored, i.e., in a closed container due to the partial pressure of pyridinium salt around the microcapsules. The compound is chemically stable until penetrating the capsule wall during storage and after application.

Suitable fill stabilizers absorb ultraviolet light in the range of about 270-350 nm and convert it into a harmless form. They have high absorption coefficients (eg, log molar extinction coefficients) in the vicinity of the spectrum's ultraviolet light, but minimal absorption near the visible light of the spectrum. They show no substantial chemical reaction with the isocyanate groups and primary amine groups of the shell forming compounds during the microencapsulation process Compounds which can be used as fill stabilizers are 2,4-dihydroxy benzophenone, 2-hydroxy-4-methoxy benzo Substituted benzophenones such as phenone, 2-hydroxy-4-octyloxy benzophenone, etc. 2- (2-hydroxy-5'-methylphenyl) benzotriazole, 2- (3 ', 5'-diallyl- Benzotriazoles such as 2'-hydroxyphenyl) benzotriazole, etc., ethyl 2-cyano-3,3-diphenyl acrylate, 2-ethylhexyl-2-cyano-3,3-diphenyl acetate, Substituted acrylates such as phenyl salicyl Salicylates such as nitrate, 5-butyl phenyl salicylate, etc., and nickel organic compounds such as nickel bis (octylphenol) sulfide, etc. Additional examples of charge stabilizers of this kind include Kirk-Othmer, It can be found in the Encyclopedia of Chemical Technology The charge stabilizer constitutes up to 5% by weight of the microcapsule composition and generally constitutes 0.01 to 2%.

Another embodiment of the present invention may include heat sensitive materials that promote storage stability, in particular in light resistance, and the microcapsules have an ultraviolet absorber enclosed therein, which may be applied in various fields. Preferred components that may be present in the base material include materials capable of absorbing heat and protecting the underlying material from overheating. Thermal energy is absorbed by the phase change of such materials without causing an increase in the temperature of these materials. Suitable phase change materials are paraffinic hydrocarbons, ie straight chain hydrocarbons represented by the formula C _n H _{n +2} where _n can be in the range from 13 to 28. Other compounds suitable for phase change materials include PABA salts, 2,2-dimethyl-1,3-propane diol (DMP), 2-hydroxymethyl-2-methyl-1,3-propane diol (HMP) and the like Compound. Also useful are fatty acid esters, such as methyl palmitate. Phase change materials that can be used include paraffinic hydrocarbons.

Thermal sensitivity using color-forming reactions between colorless or pale basic dyes and organic or inorganic color acceptors to contact the two chromogenic materials with each other by heat to obtain a record image. Recording materials are well known. Such heat sensitive recording materials are adapted for use with relatively inexpensive, compact and easy-to-maintain recording devices, and therefore, record media for facsimile systems, various computers, and the like. recording media) has a wide range of applications. To improve the light resistance of the heat sensitive recording material, finely separated ultraviolet absorbers or blocking agents can be added to the heat sensitive recording layer or protective layer.

Additional embodiments of the present invention provide microcapsules that can retain ultraviolet absorbents, exhibit resistance to bursting at conventional pressures, and / or have suitable ultraviolet absorption efficiencies.

Embodiments of the present invention may include a substrate, a recording layer formed on the substrate and containing a colorless or pale basic dye and a color receptor, and a protective layer formed on the recording layer, wherein the recording material is formed therein. A microcapsule having an ultraviolet absorber enclosed in and having substantially no color forming ability may include a heat sensitive recording material characterized in that it is contained in the protective layer.

The present invention also provides microcapsules having an ultraviolet absorber and, if necessary, an organic solvent enclosed therein, wherein the capsule has a capsule wall film composed of synthetic resin and an average particle size of about 0.1 to 3 μm.

Below are examples of ultraviolet absorbers that can be used in the present invention. Salicylic acid type ultraviolet absorbers such as phenyl salicylate, p-tert-butylphenyl salicylate, p-octylphenyl salicylate and its equivalents; 2,4-dihydroxybenzophenone, 2-hydroxy-4-methoxybenzophenone, 2-hydroxy-4-octyloxybenzophenone, 2-hydroxy-4-dodecyloxybenzophenone, 2,2 '-Dihydroxy-4-methoxybenzophenone, 2,2'-dihydroxy-4,4'-dimethoxybenzophenone, 2-hydroxy-4-methoxy-5-sulfobenzophenone and its equivalents Benzophenone type ultraviolet absorbers such as; Cyanoacrylate type ultraviolet absorbers such as 2-ethylhexyl 2-cyano-3,3-diphenyl-acrylate, ethyl 2-cyano-3,3-diphenylacrylate and their equivalents; Bis (2,2,6,6-tetramethyl-4-piperidyl) sebacate, bis (2,2,6,6-tetramethyl-4-piperidyl) succinate, bis (l, 2, Steric hindered amines such as 2,6,6-pentamethyl-4-piperidyl) 2- (3,5-di-tert-butyl-4-hydroxybenzyl) -2-n-butyl malonate and its equivalents hindered amine type ultraviolet absorbers; 2- (2'-hydroxyphenyl) benzotriazole, 2- (2'-hydroxy-5'-methylphenyl) benzotriazole, 2- (2'-hydroxy-5-tert-butylphenyl) benzotria Sol, 2- (2'-hydroxy-3 ', 5'-di-tert-butylphenyl) benzotriazole, 2- (2'-hydroxy-3'-tert-butyl-5'-methylphenyl)- 5-chlorobenzotriazole, 2- (2'-hydroxy-3 ', 5'-di-tert-butylphenyl) -5-chlorobenzotriazole, 2- (2'-hydroxy-3', 5 '-Di-tert-butylphenyl) -5-tert-butylbenzotriazole, 2- (2'-hydroxy-3', 5'-di-tert-amylphenyl) benzotriazole, 2- (2 ' -Hydroxy-3 ', 5'-di-tert-amylphenyl) -5-tert-amylbenzotriazole, 2- (2'-hydroxy-3', 5'-di-tert-amylphenyl)- 5-methoxybenzotriazole, 2- [2'-hydroxy-3 '-(3 ", 4", 5 ", 6" -tetrahydrophthalimido-methyl) -5'-methylphenyl] benzotriazole 2- (2'-hydroxy-5'-tert-octylphenyl) benzotriazole, 2- (2'-hydroxy-3'- sec-butyl-5'-tert-butylphenyl) benzotriazole, 2- (2'-hydroxy-3'-tert- Mill-5'-phenoxyphenyl) -5-methylbenzotriazole, 2- (2'-hydroxy-5'-n-dodecylphenyl) benzotriazole, 2- (2'-hydroxy-5 ' -Cec-octyloxyphenyl) -5-phenylbenzotriazole, 2- (2'-hydroxy-3'-tert-amyl-5'-phenylphenyl) -5-methoxybenzotriazole, 2- [2 Benzotriazole type ultraviolet absorbents which are solid at conventional temperatures such as '-hydroxy-3', 5'-bis (α, α-dimethylbenzyl) phenyl] benzotriazole and its equivalents; 2- (2'-hydroxy-3'-dodecyl-5'-methylphenyl) -benzotriazole, 2- (2'-hydroxy-3'-undecyl-5'-methylphenyl) -benzotriazole, 2- (2'-hydroxy-3'-tridecyl-5'-methylphenyl) -benzotriazole, 2- (2'-hydroxy-3'- tetradecyl-5'-methylphenyl) -benzotriazole, 2- (2'-hydroxy-3'-pentadecyl-5'-methylphenyl) -benzotriazole, 2- (2'-hydroxy-3'-hexadecyl-5'-methylphenyl) -benzotriazole, 2- [2'-hydroxy-4 '-(2 "-ethylhexyl) oxyphenyl] -benzotriazole, 2- [2'-hydroxy-4'-(2" -ethylheptyl) oxyphenyl]- Benzotriazole, 2- [2'-hydroxy-4 '-(2 "-ethyloctyl) oxyphenyl] -benzotriazole, 2- [2'-hydroxy-4'-(2" -propyloctyl) Oxyphenyl] -benzotriazole, 2- [2'-hydroxy-4 '-(2 "-propylheptyl) oxyphenyl] -benzotriazole, 2- [2'-hydroxy-4'-(2" -Propylhexyl) oxyphenyl] -benzotriazole, 2- [2'-hydroxy-4 '-(l "-ethylhexyl) oxyphenyl] -benzotriazole, 2- [2'-hydroxy-4' -(1 "-ethylheptyl) oxyphenyl ] -Benzotriazole, 2- [2'-hydroxy-4 '-(1 "-ethyloctyl) oxyphenyl]-benzotriazole, 2- [2'-hydroxy-4'-(1" -propyl Octyl) oxyphenyl] -benzotriazole, 2- [2'-hydroxy-4 '-(1 "-propylheptyl) oxyphenyl] -benzotriazole, 2- [2'-hydroxy4'-(1 "-Propylhexyl) oxyphenyl] -benzotriazole, 2- (2'-hydroxy-3'- sec-butyl-5'-tert-butylphenyl-5-n-butylbenzotriazole, 2- (2 '-Hydroxy-3'-cec-butyl-5'-tert-butylphenyl) -5-tert-pentyl-benzotriazole, 2- (2'-hydroxy-3'-cec-butyl-5'- Tert-butylphenyl) -5-n-pentyl-benzotriazole, 2- (2'-hydroxy-3'- sec-butyl-5'-tert-pentylphenyl) -5-tert-butylbenzotriazole, 2- (2'-hydroxy-3'-sec-butyl-5'-tert-pentylphenyl) -5-n-butylbenzotriazole, 2- (2'-hydroxy-3 ', 5'-di -Tert-butylphenyl) -5-cec-butylbenzotriazole, 2- (2'-hydroxy-3 ', 5'-di-tert-pentylphenyl) -5-cec-butylbenzotriazole, 2- (2'-hydroxy-3'-tert-butyl-5'-tert- Pentylphenyl) -5-cec-butylbenzotriazole, 2- (2'-hydroxy-3 ', 5'-di-cec-butylphenyl) -5-chlorobenzotriazole, 2- (2'-hydrate Roxy-3 ', 5'-di-sec-butylphenyl) -5-methoxybenzotriazole, 2- (2'-hydroxy-3', 5'-di-sec-butylphenyl) -5-tert -Butylbenzotriazole, 2- (2'-hydroxy-3 ', 5'-di-sec-butylphenyl) -5-n-butylbenzotriazole, octyl 5-tert-butyl-3- (5- Chloro-2H-benzotriazol-2-yl) -4-hydroxybenzene-propionate, methyl 3- [3-tert-butyl-5- (2H-benzotriazol-2-yl) -4-hydrate A benzotriazole type ultraviolet absorbent which is liquid at normal temperatures, such as condensate of oxyphenyl] propionate and polyethylene glycol (molecular weight: about 300) and its equivalents. Of course, ultraviolet absorbers are not limited to those listed above and may be used in a mixture of two or more of them if necessary.

The amount of the ultraviolet absorber used is not particularly limited, but the amount may be adjusted to 10 to 500 parts by weight, and generally 20 to 250 parts by weight.

Microcapsules used in the present invention can be prepared by a variety of known methods. They emulsify and disperse a core material (oily liquid) comprising an ultraviolet absorber and, if necessary, an organic solvent in an aqueous medium, and a wall film of high-molecular weight material around the resulting oily droplets. It is prepared by the step of forming.

Examples of useful high-molecular weight materials that form wall films of microcapsules include polyurethane resins, polyurea resins, polyamide resins, polyester resins, polycarbonate resins, aminoaldehyde resins, melamine resins, polystyrene resins, styrene- Acrylate copolymer resins, styrene-methacrylate copolymer resins, gelatin, polyvinyl alcohol, and the like. In particular, microcapsules with wall films of polyurea resins, polyurethane resins and aminoaldehyde resins, rather than synthetic resins, in particular other resins, have excellent retainability and high thermal resistance of ultraviolet absorbers and thus thermal heads. It shows an excellent additional effect of providing the function of the pigment to be embedded in the protective layer to prevent sticking of the furnace. In addition, microcapsules with wall films of polyurea resins or polyurethane resins have a lower refractive index than other capsules and microcapsules with conventional pigments, which are spherical in shape and are therefore advantageously available, which in large quantities in the protective layer If present, the density of the recorded image will not be reduced (so-called whitening) because of irregular reflection of light. In addition, polyurea resins and polyurethane resins are more elastic than aminoaldehyde resins and therefore polyurea resins and polyurethane resins are generally used as wall films for microcapsules used under high pressure conditions. On the other hand, microcapsules with wall films made of aminoaldehyde resins have the advantage that the thickness of the wall film can be controlled regardless of the particle size of the emulsion, which means that the microcapsules can This can be produced by addition.

The present invention may include an organic solvent together with the ultraviolet absorbent. The organic solvent is not particularly limited and various hydrophobic solvents used in the field of pressure sensitive manifold paper can be used. Examples of organic solvents include phosphates such as tricresyl phosphate and octyldiphenyl phosphate, phthalates such as dibutyl phthalate and dioctyl phthalate, carboxylates such as butyl oleate, various fatty acid amides, diethylene glycol dibenzoate, mono Alkylated naphthalenes such as isopropylnaphthalene and diisopropylnaphthalene, 1-methyl-1-phenyl-1-tolylmethane, 1-methyl-1-phenyl-1-xylylmethane, 1-phenyl Alkylated benzenes such as -1-tolylmethane, alkylated biphenyls such as isopropylbiphenyl, acrylates such as trimethylolpropane triacrylate, esters of polyols and unsaturated carboxylic acids, chlorinated paraffins and kerosine. These solvents can be used individually or in mixtures of two or more of them. Among such hydrophobic media with high boiling points, tricresyl phosphate and 1-phenyl-1-tolylmethane are preferred because they show high solubility in relation to the ultraviolet absorbers used in the present invention. In general, the lower the viscosity of the core material, the smaller the size of the particles obtained from emulsificaiton and the narrower the particle size distribution, so that solvents having a lower boiling point can be used together to lower the viscosity of the core material. Examples of such low boiling solvents are ethyl acetate, butyl acetate, methylene chloride and the like.

The amount of the organic solvent used may be appropriately adjusted depending on the kind of ultraviolet absorber used and the kind of organic solvent, and is not particularly limited. For example, when using the ultraviolet absorber which is liquid at normal temperature, an organic solvent is not necessarily used. However, when using a ultraviolet absorber that is solid at room temperature, since the ultraviolet absorber is preferably completely dissolved in the microcapsules, the amount of the organic solvent is, for example, in the case of microcapsules of polyurea resin or polyurethane resin. And generally adjusted to about 10 to 60 weight percent, or about 20 to 60 weight percent, based on the total amount of organic solvent, ultraviolet absorber and wall-forming material. In addition, for microcapsules of aminoaldehyde resins, the amount of organic solvent is usually adjusted to about 50 to 2000%, generally about 100 to 1000% by weight of the ultraviolet absorber.

In addition, an absorber may be used. It does not interfere with exposure at other wavelengths (inactive regions) of the microcapsules without excessively lowering the sensitivity of the microcapsules in the spectral sensitivity range (active region) where the microcapsules are intended to be exposed. Absorbents that reduce the sensitivity of the microcapsules in the spectral sensitivity range should be selected. In some cases, it may be necessary to match the absorbent properties of the absorbent in the active and inactive areas to achieve optimal exposure properties. Generally, absorbents with absorbance coefficients of greater than about 100 / M in the inactive region of the microcapsules and less than about 100,000 / M cm in the active region are used. If the absorbent is incorporated directly into the photosensitive composition, ideally the absorbent should not inhibit free radical polymerization and should not produce free radicals upon exposure.

The absorbent used in the present invention may be selected from those known in the photographic art. Examples of such compounds are dyes commonly used as silver halide sensitizing dyes in color photography (eg, cyanine, merocyanine, hemicyanine and styryl pigments) and ultraviolet light. And absorbents. Many colored dyes that absorb outside the desired sensitivity region of the microcapsules and do not absorb strongly within the region can also be used as absorbers in the present invention. Of these, Sudan I, Sudan II, Sudan HI, Sudan Orange G, Oil Red O, Oil Blue N, and Fast Garnet GBC are examples of potentially useful compounds.

Additionally, ultraviolet absorbers which may be preferred include those selected from hydroxybenzophenone, hydroxyphenylbenzo-triazole and formamidine. Absorbents can be used alone or in combination to achieve the desired spectral sensitivity properties.

Representative examples of useful hydroxybenzophenones are 2-hydroxy-4-n-octoxybenzophenone (UV-CHEK AM-300 from Ferro Chemical Division, Mark 1413 from Argus Chemical Division, Witco Chem. Corp., and Cyasorb UV -531 Light Absorber from American Cyanamid, 4-dodecyl-2-hydroxybenzophenone (Eastman Inhibitor DOBP from Eastman Kodak), 2-hydroxy-4-methoxybenzophenone (Cyasorb UV-9 Light Absorber from American Cyanamid ), And 2,2'-dihydroxy-4-methoxybenzophenone (Cyasorb UV-24 Light Absorber from American Cyanamid). Representative examples of useful hydroxybenzophenyl benzotriazoles are Tinuvin P from Ciba-Geigy Additives Dept, 2- (3 ', 5'-di Tert-butyl-2'hydroxyphenyl) -5-chlorobenzotriazole (Tinuvin 327 from Ciba-Geigy), and 2- (2-hydroxy-5-t-octylphenyl) benzotriazole (Cyasorb UV-5411 Light Absorber from American Cyanamid). Representative examples of useful formamidines are disclosed in US Pat. No. 4,021,471 and described as N- (p-ethoxy-carbonylphenyl) -N'-ethyl-N'-phenylformamidine (Givsorb UV-2 from Givaudan Corp. Including.) The optimal absorbent and concentration of absorbent for a particular application depends on the absorption maximum and absorbance coefficient of the absorbent candidate and the spectral sensitivity properties of the associated photoinitiator.

Additionally, microcapsules, photosensitive compositions, image-forming agents, developers, and developing techniques are described in US Pat. Nos. 4,399,209 and 4,440,846.

In particular, formulations to be applied in the form of spraying, such as water disperable concentrates or wettable powders, may be used for the condensation of surfactants such as wetting and dispersing agents, for example formaldehyde and nattalene sulfonate. Products, alkylarylsulfonates, lignin sulfonates, fatty acid alkyl sulphates, and ethoxylated alkylphenols and ethoxylated fatty alcohols.

2. Liposomes  Formulations ( Liposomal Formulation )

As used herein, the term "liposome" refers to a structure comprising a lipid bilayer surrounding one or more aqueous compartments. Walls are made from lipid molecules that tend to form bilayers and minimize their surface area. The lipid molecules that make up liposomes have hydrophilic and lipophilic moieties. Upon exposure to water, the lipid molecules form a bilayer membrane, in which the lipid end of the molecule is towards the center of the membrane, and the opposing polar ends form the inner and outer surfaces of the bilayer. Thus, each side of the molecule provides a hydrophilic surface, and the interior of the molecule constitutes a lipophilic medium.

Liposomes can be classified into several categories based on the overall size and properties of the lamellar structure. The classification of liposomes includes small unilamellar vesicles (SUV), multilamellar vesicles (MLV), large unilamellar vesicles (LUV), and oligo lamellar vesicles. It includes. SUVs may comprise a single lipid bilayer about 20-50 nanometers in diameter and surrounding an aqueous compartment. The characteristic of the SUV is that a large amount of total lipid, about 70%, is located in the outer layer of the bilayer. SUVs are single compartment vesicles of fairly uniform size, but MLVs vary widely in diameter, up to about 30,000 nanometers, and liposome bilayers are typically structured, with the closed concentric lamellae separating the lamellae from the next lamellae. lamellae) and multicompartmental. Large single lamellae vesicles are referred to as large lamellae vesicles because of their large diameters ranging from about 600 nanometers to 30 microns. Oligo lamellar vesicles are intermediate liposomes with an aqueous space greater than MLV and an aqueous space less than LUV. Oligo lamellae vesicles have more than one inner compartment and several concentric lamellae, but generally have a smaller number of lamellae than the MLV.

Various methods of preparing liposomes are known in the art, some of which are described in Liposome Technology (Gregoriadis, G., editor, three volumes, CRC Press, Boca Raton 1984) or by Lichtenberg and Barenholz. Methods of Biochemical Analysis, Volume 33, 337-462 (1988). Additional methods for preparing liposome formulations are described in US Pat. No. 7,022,336; No. 6,989,153; No. 6,726,924; No. 6,355,267; 6,110,491; 6,007,838; 6,007,838; 5,094,785 and 4,515,736. Liposomes are also recognized to be useful for encapsulating biologically active substances. In particular, methods of preparation involving encapsulation of DNA by liposomes, and methods having direct application to liposome-mediated transfection, are described by Hug and Sleight in Biochimica and Biophysica Acta, 1097, 1-17 ( 1991).

Liposomes of the invention can be prepared from phospholipids, but other molecules of similar molecular shape and size with both hydrophobic and hydrophilic moieties can be used. For the purposes of the present invention, all such liposome-forming molecules will be referred to herein as lipids. One or more natural and / or synthetic lipid compounds may be used in the preparation of liposomes.

Representative suitable phospholipids or lipid compounds that form the first liposomes useful in the present invention include phosphatidylcholine (lecithin), lysocithin, lysophosphatidylethanol-amine, phosphatidylserine, phosphatidylinositol, sphingomyelin, phosphatidylethanolamine (cephalin), carboxyl Phospholipid-related substances such as diolipine, phosphatidic acid, cerebroside, dicetylphosphate, phosphatidylcholine, and dipalmitoyl-phosphatidylglycerol. Additional nonphosphorous-containing lipids include stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl sterate, isopropyl myristate, amphoteric acrylic polymers, Fatty acids, fatty acid amides, cholesterol, cholesterol esters, diacylglycerols, diacylglycerolsuccinates, and equivalents.

As will be appreciated by those skilled in the art, different lipids having different properties, cationic, anionic or neutral may be used, however the methods of preparation may be the same, regardless of the lipid combination used. More specifically, once the lipids to be used in the liposomes are selected, they can be dissolved in organic solvents for complete mixing. The organic solvent can be removed by evaporation and subsequent drying leaving a lipid film of uniform lipid mixture. The lipid mixture can be frozen and dried with a cake. The lipid cake can be stored frozen until hydrated.

Addition of an aqueous medium and stirring of the vessel hydrate the lipid cake. The resulting product is a large multilayer lamellae vesicle. This structure may comprise concentric circles of lipid bilayer separated by water. Large multi-lamellar vesicles can be miniaturized by the application of energy in the form of mechanical energy in the extrusion process or in the form of sonic energy in sonication. Hydrated lipids can be processed through a polycarbonate filter with progressively smaller pores to produce particles with a diameter of similar size to the pores. Before the final pore size is used, the lipid suspension is subjected to several freeze-thaw cycles to make the final particle uniform. Final particle size depends in part on the lipid combination used. Average particle size can be reproduced between batches. This process can produce large monolamellar vesicles that can be reduced to small monolamellar vesicles by the application of sonic energy from a sonicator. Particles in the test tube that are sonicated can be removed by centrifugation. The average size of the resulting vesicles can be influenced by the composition, concentration, capacity and temperature of the lipid mixture and the duration, power, and tuning of the sonicator.

Certain liposome preparation methods include hand-shaken methods, sonication methods, reverse-phase evaporation methods, freeze-dry rehydration methods, and detergent depletion. Including but not limited to methods. According to the hand-shaken method, lipid molecules are introduced into an aqueous environment to produce liposomes. When the dried lipid film is hydrated, the lamellae swell into the myelin figure. Mechanical agitation, such as vortexing, shaking, swirling or pipetting, breaks the myelin form (thin lipid tubule) and recombines exposed hydrophobic edges ( reseal) resulting in the formation of liposomes.

Sonication methods can be used to produce small monolamellar vesicles. Two representative sonication techniques include probe sonication and bath sonication. During probe sonication, the tip of the sonicator is directly immersed in the liposome dispersion. Dissipation of energy at the tip can result in local overheating, and thus the container can be dipped into an ice / water bath. During solution sonication, liposome dispersions in the tubes are placed in the solution sonicator. The material to be sonicated, unlike the probe unit, may be kept in a sterile container or under an inert atmosphere.

The reverse-phase evaporation method is based on the formation of inverted micelles. More specifically, inverted micelles are formed upon sonication of a mixture of a buffered aqueous phase comprising a water soluble molecule to be encapsulated into liposomes and an organic phase in which amphiphilic molecules are solubilized. Slow removal of the organic solvent converts this inverted micelle into a viscous state similar to the gel. During some point in this procedure, the gel state collapses and some of the inverted micelles disintegrate. Excess phospholipids in the environment contribute to the formation of a complete bilayer around the remaining micelles, which leads to the formation of liposomes. Liposomes prepared by reverse-phase evaporation methods can be prepared from a variety of lipid formulations and have an aqueous volume-to-lipid ratio four times higher than multilayer lamellar liposomes or hand-shaken liposomes. There is a tendency.

During the freeze-drying rehydration method, freeze-dried liposomes are formed from preformed liposomes. During dehydration, the material to be encapsulated into the lipid bilayer and liposomes is in intimate contact. During reswelling, the likelihood of encapsulation of attached molecules is increased. The aqueous phase is generally added in small amounts to the dry matter. In general, the total dose used for rehydration is less than the starting dose of the liposome dispersion.

Detergent exhaustion methods can be used for various liposomes and proteoliposome preparations. Detergents can be exhausted from mixed detergent-lipid micelles by various techniques resulting in the formation of homogeneous liposomes. Indeed, lipids at temperatures below their phase transition temperature can be used in this preparation method. However, not all detergents are suitable for this method. Representative detergents include, but are not limited to, sodium cholate, alkyl (thio) glucosides, and alkyloxypolyethylenes. Mixed micelles are prepared by adding a concentrated detergent solution to the multilayered lamellar liposomes (the final concentration of detergent should be much higher than the critical micelle concentration (CMC) of detergent). The use of different detergents can result in different size distributions of the formed vesicles. Faster burnout rates can produce smaller size liposomes. The use of different detergents can also result in different ratios of large monolamellar vesicles / oligolamellae vesicles / multilayer lamellae vesicles.

3. Michelle ( micelle ) And vissel ( bicelle )

As used herein, "micelle" means an aggregate of surfactant molecules dispersed in a liquid colloid. The micelles may be spherical in shape but may exist in other forms, including but not limited to oval, cylindrical, bilayer and vesicles. The shape of the micelle can be controlled primarily by the molecular shape of the surfactant molecule; The form also depends on the conditions (eg, temperature or pH, and the type and concentration of salt added). In micelles, the hydrophobic tails of several surfactant molecules are assembled into an oil-like core with less contact with water. In contrast, surfactant monomers are surrounded by water molecules that form a "cage" of molecules connected by hydrogen bonds. In nonpolar solvents, hydrophilic groups form the core of a micelle, and hydrophobic groups remain on the surface of the micelle (so-called reverse micelle).

Micelles can be formed when the concentration of the surfactant is higher than the critical micelle concentration (CMC) and the temperature of the system is higher than the critical micelle temperature or the kraft temperature. In water, despite the fact that aggregation of surfactant molecules together reduces entropy, the hydrophobic effect is the driving force for micelle formation. In general, at concentrations above CMC, the entropy penalty of forming aggregates of surfactant molecules is less than the entropy penalty of water molecules forming cages.

As used herein, "bicelle" refers to bilayered mixed micelles. Vicels are characterized by a mixture of lipids of phospholipids forming short-chain bilayers and detergents forming short-chain micelles.

The preparation of micelles and bicells is well known in the art. More details regarding the fabrication of these structures are described in US Pat. No. 6,897,297; 6,696,081; 6,696,081; No. 6,586,559; No. 6,444,793; And 5,534,259.

4. Peg ( PEGylation )

Attachment of polyalkylene moieties as described herein to increase water solubility or solubility in aqueous solutions and / or to extend the half-life of the native compounds discussed herein. Can be used. Any conventional PEGylation method can be used as long as the PEGylated agent maintains the desired activity. See also Schacht, E.H. et al. See Poly (ethylne glycol) Chemistry and Biological Applications, American Chemical Society, San Francisco, CA 297-315 (1997).

Polyalkylene glycol is a biocompatible polymer, and as used herein, polyalkylene glycol means a straight or branched polyalkylene glycol polymer such as polyethylene glycol, polypropylene glycol and polybutylene glycol, And monoalkyl ethers of polyalkylene glycols. In some embodiments of the present invention, the polyalkylene glycol polymer is a lower alkyl polyalkylene moiety such as polyethylene glycol moiety (PEG), polypropylene glycol moiety, or polybutylene glycol moiety. PEG has the formula -H (CH2CH2O) _n H, _wherein n is from about 1 to about 4000 or more. In some embodiments, n is 1 to 100, and in other embodiments, n is 5 to 30. PEG can have an average molecular weight ranging from about 1 to about 22,000. For example, an average molecular weight of about 300 corresponds to a case where n is 5, an average molecular weight of about 2,300 corresponds to a case where n is 50, and an average molecular weight of 13,300 corresponds to a case where n is 300 and about 22,000 The average molecular weight of N corresponds to the case where n is 500. In some embodiments, the PEG moiety can be straight or branched. In other embodiments, PEG may be attached to a group such as hydroxyl, alkyl, aryl, acyl or ester. In some embodiments, PEG may be an alkoxy group, one terminal being a relatively inert, and the other terminal may be an alkoxy PEG, for example methoxy-PEG (or mPEG), which is a hydroxy group. PEG is a commercially available product that can be synthesized or can be readily obtained.

According to some embodiments of the invention, the PEGylated compounds of the invention are water soluble, soluble in isopropyl alcohol (IPA), ethanol (ETOH), dimethyl sulfoxide (DMSO) and methanol (MEOH) and non-peptide It is less sensitive to UV light than the counterpart counterpart, and / or the synthesis is economical.

The compounds of the present invention may be PEGylated at four or more sites and / or PEGylated with many different PEG lengths and molecular weights. In some embodiments, the PEG moiety is PEG ₂₀₀ to PEG _500O . PEGylated compounds of the present invention may also be characterized by increased solubility, increased stability, lower toxicity, reduced degradation and chemical sensitivity, and / or similar molecules and other molecules such as known drugs, biological tags ( increased conjugation potential to biological tags, labels, fluorescence, radioisotopes and their equivalents.

Embodiments of the invention include assays in which the sample is combined with a labeling reagent. A "labeling reagent" is a luminescent macromolecule comprising a chimeric formed from a luminescently labeled macromolecule, a green fluorescent protein, and a mutant thereof, including a fluorescent protein analog and a biosensor. Luminescent macromolecular chimera, luminescent labeled primary or secondary antibodies that react with cellular antigens involved in physiological reactions, luminescent stains, dyes, and other small molecules.

By "biosensor" is meant a macromolecule consisting of a luminescent probe or probes that reports a biological functional domain and environmental changes occurring internally or on their surface. A series of luminescent labeled macromolecules designed to detect and report such changes was referred to as "fluorescent-protein biosensors." The protein component of the biosensor provides an evolved molecular recognition moiety. Fluorescent molecules attached to protein components in the vicinity of the active site convert environmental changes into fluorescent signals that can be detected using a suitable temporal and spatial resolution system. Because the modulation of circular protein activity in living cells is reversible, and fluorescent protein biosensors can be designed to detect reversible changes in protein activity, these biosensors are inherently reusable.

"High content screening (HCS)" refers to signaling pathways and apoptosis, cell division, cell adhesion, migration, exocytosis, cell growth and metabolism, protein synthesis, enzyme function, cell-cells It can be used to measure the effects of drugs on complex molecular events such as, but not limited to, cell-cell communication and detection of healthy and / or diseased cells. Multicolor fluorescence allows multiple target and cellular processes to be analyzed on a single screen. Cross-correlation of cellular responses will generate a large amount of information needed for target validation and lead optimization.

Methods for screening cells treated with dyes and fluorescent reagents are well known in the art. As reporter molecules, there is a significant amount of literature relating to the genetic manipulation of cells to produce fluorescent proteins, such as modified green fluorescent proteins (GFP). Some properties of wild type GFP are disclosed by Morise et al. (Biochemistry 13 (1974), p. 2656-2662), and Ward et al. (Photochem. Photobiol. 31 (1980), p. 611-615). The jellyfish Aequorea Victoria's GFP has an excitation maximum at 395 nm and an emission maximum at 510 nm and does not require any extraneous factors for fluorescence activity. The use of the GFP disclosed in the literature varies and studies of gene expression and protein localization (Chalfie et al., Science 263 (1994), p. 12501-12504), visualization of subcellular organelles Sudan (Rizzuto et al., Curr. Biology 5 (1995), p. 635-642), visualization of protein transport along the secretory pathway (Kaether and Gerdes, FEBS Letters 369 (1995), p. 267-271)) Plant cells (Hu and Cheng, FEBS Letters 369 (1995), p. 331-334) and Drosohila embryos (Davis et al., Dev. Biology 170 (1995), p. 726-729) Expression, and use as reporter molecules fused to another protein of interest. Similarly, WO 96/23898 relates to a method for detecting biologically active substances that affect intracellular processes by using GFP constructs with protein kinase activation sites. This patent and all other patents referenced in this application are incorporated herein by reference in their entirety.

Numerous references relate to GFP proteins in biological systems. For example, WO 96/09598 discloses a system for separating target cells using expression of GFP-like proteins. WO 96/27675 discloses expression of GFP in plants. WO 95/21191 discloses modified GFP proteins expressed in transformed individuals for detecting mutagenesis. U.S. Pat.Nos. 5,401,629 and 5,436,128 disclose intracellular delivery of extracellular signals using recombinant cells comprising reporter gene constructs comprising transcriptional regulatory elements that express cell surface receptors and respond to the activity of cell surface receptors. Assays and compositions for detecting and evaluating transduction are disclosed. There are also a number of publications examining the use of GFP proteins.

Performing screening for thousands of compounds requires parallel handling and processing of multiple compounds and analyte reagents. Standard High Throughput Screen (“HTS”) is a combination of compounds and biological compounds with some indicator compounds loaded into an array of wells of standard microtiter plates with 96 or 384 wells. A mixture of reagents is used. Fluorescent luminescence, optical density, or radioactive signals, measured from each well, integrate the signals from all the materials in the wells to produce an overall population average of all the molecules in the wells.

Another system, the fluorescence imaging plate reader (FLEPR), has a low angle to selectively fluoresce within about 200 microns from the bottom of the well of a standard 96 well plate to reduce the background during imaging of the cell monolayer. Low angle laser scanning illumination and masks are used. The system uses a charge couple device (CCD) camera to image the entire area of the plate bottom. This system measures the signal derived from the cell monolayer at the bottom of the well, but the measured signal is averaged over the entire area of the well and is therefore considered a measure of the average response of the cell population. Images are analyzed to calculate total fluorescence per well in cell-based assays. Fluid delivery devices have also been incorporated into cell-based screening systems, such as the FLIPR system, to initiate the reaction, which is observed as an overall cell population mean response using a macro-imaging system.

In contrast to High Throughut Screening, a variety of high-content screenings (HCS) are available to address the need for more detailed information about the temporal-spatial dynamics of cellular constituents and processes. Developed. High-content screening automates the extraction of multicolor fluorescence information derived from specific fluorescence-based reagents embedded into cells (Giuliano and Taylor (1995), Curr. Op. Cell Biol. 7: 4; Giuliano et al. 1995) Ann. Rev. Biophys. Biomol. Struct. 24: 405). Cells are analyzed using an optical system capable of measuring temporal and spatial dynamics (Farkas et al. (1993) Ann. Rev. Physiol. 55: 785; Giuliano et al. (1990) In Optical Microscopy for Biology. B. Herman and K. Jacobson (eds.), Pp. 543-557.Wieley-Liss, New York; Hahn et al (1992) Nature 359: 736; Waggoner et al. (1996) Hum.Pathol. 27: 494 ). The concept is to treat each cell as a "well" with temporal and spatial information about the activity of the labeled components.

The types of biological and molecular information accessible through fluorescence-based reagents applied to cells include ion concentration, membrane potential, specific translocation, enzyme activity, gene expression, and metabolites, proteins, lipids, carbohydrates, and nucleic acid sequences. The presence, amount, and pattern of (DeBiasio et al., (1996) Mo I. Biol. Cell. 7: 1259; Giuliano et al., (1995) Ann. Rev. Biophys. Biomol. Struct. 24: 405; Heim and Tsien, (1996) Curr. Biol. 6: 178).

High-content screening can be performed in immobilized cells using fluorescently labeled antibodies, biological ligands and / or nucleic acid hybridization probes, or in living cells using multicolor fluorescent indicators and “biosensors”. The choice of fixed or live cell screening depends on the particular cell-based assay required.

As will be appreciated by those skilled in the art to which the present invention pertains, any of the methods and screenings described above may be used with the compounds of the present invention.

In general, fixed cell assays are the simplest, in which the array of living cells is first treated with various compounds and dosages in a microtiter plate format, and then the cells are fixed, labeled with specific reagents, and quantified. Because it can be. After fixation no environmental control of the cells is required. Spatial information is obtained, but only at one time point. The availability of thousands of antibodies, ligands and nucleic acid hybridization probes that can be applied to cells makes this method an attractive approach to various kinds of cell-based screening. The fixation and labeling steps can be automated, allowing for efficient processing of the assay.

Living cell assays are more sophisticated and powerful because arrays of living cells containing the desired reagents can be screened not only spatially, but also over time. During the measurement, the environmental control (temperature, humidity, and carbon dioxide) of the cell must be maintained because the physiological health of the cell must be maintained for multiple fluorescence measurements over time. There is an increasing list of fluorescent physiological indicators and "biosensors" that can report changes in biochemical and molecular activity in cells (Giuliano et al., (1995) Ann. Rev. Biophys. Biomol. Struct. 24: 405; Hahn et al., (1993) In Fluorescent and Luminescent Probes for Biological Activity.WT Mason, (ed.), Pp. 349-359, Academic Press, San Diego.

The availability and use of fluorescence-based reagents has contributed to the early development of high-content screening of fixed and living cells. Advances in equipment for automatically extracting multi-color, high-content information have enabled HCS to be developed as an automated tool. Taylor Scientist 80 (1992), p. 322-335, describes a number of such methods and their applications. For example, Proffitt et al. (Cytometry 24: 204-213 (1996)) describe a semi-automatic method for measuring relative cell numbers in situ in various tissue culture plate formats, particularly in 96-well microtiter plates. (semi-automated) fluorescence digital imaging systems are described. The system consists of an epifluorescence inverted microscope with a motorized stage, a video camera, an image intensifier, and a microcomputer with a PC-vision digitizer. Turbo Pascal software controls the stage and scans the plate to capture multiple images per well. The software calculates total fluorescence per well, provides daily calibration, and easily sets up various tissue culture plate formats. Thresholding of the fluorescence reagent and digital image only when taken by living cells is used to reduce background fluorescence without removing excess fluorescent reagent.

Scanning confocal microscope imaging (Go et al., (1997) Analytical Biochemistry 247: 210-215; Goldman et al., (1995) Experimental Cell Research 221: 311-319) and multiquantum microscope imaging (multiphoton microscope imaging) (Denk et al., (1990) Science 248: 73; Gratton et al., (1994) Proc. of the Microscopical Society of America, pp. 154-155) are also microscopic samples. It is an established method of obtaining high resolution images of. The main advantage of this optical system is its low depth of focus, which allows a feature of a limited axial extent to form an image against the background. For example, it is possible to isolate and identify the internal cytoplasmic features of attached cells from the features on the cell surface. Since scanning multiphoton imaging uses a short duration pulsed laser system to achieve the required high photon flux, fluorescence lifetime can also be measured in this system (Lakowicz et al. , (1992) Anal.Biochem. 202: 316-330; Gerrittsen et al. (1997), J. of Fluorescence 7: 11-15)), providing additional capabilities for different detection modes. Currently, small, reliable and relatively inexpensive laser systems are available, such as laser diode pumped lasers, which allow a multiphoton confocal microscope to be applied in a fairly routine manner.

The main components of the novel drug discovery paradigm are the continuous increase in the fluorescence and luminescence reagents used to measure the temporal and spatial distribution, content, and activity of intracellular ions, metabolites, macromolecules and organelles. Family. These types of reagents include labeling reagents that measure the distribution and amount of molecules in living and immobilized cells, environmental indicators that report signal transduction events in time and space, and fluorescence for measuring target molecule activity in living cells. Protein biosensors. The multiparameter approach to integrating multiple reagents in one cell can be a powerful tool for drug discovery.

The methods of the present invention include the high affinity of fluorescent or luminescent stiletto molecules and analogs for specific cellular components. Affinity for certain components can include ionic interactions, covalent bonds (including chimeric fusions with protein-based chromophores, fluorophores, and lumiphores), and hydrophobic interactions. Action, electrical potential, and in some cases, are governed by forces such as simple entrapment in intracellular components. Those skilled in the art to which this invention pertains include fluorescently labeled biomolecules, such as proteins, phospholipids, organelles, and DNA hybridization probes, which can be used for multiplexing in addition to the compounds of the invention, It will be appreciated that a wide variety of fluorescent reporter molecules are not limited thereto.

Luminescent probes are synthesized in living cells or into cells through several non-mechanical modes including diffusion, facilitative transport or active transport, signal-sequence-mediated transport, and inclusion or pinocytotic uptake. Can be transported. Mechanical bulk loading methods, well known in the art, can also be used to load luminescent probes into living cells (Barber et al. (1996), Neuroscience Letters 207: 17-20 Bright et al. (1996), Cytometry 24: 226-233; McNeil (1989) in Methods in Cell Biology, Vol. 29, Taylor and Wang (eds.), Pp. 153-173). These methods are electroporation and scrape loading, bead-loading, impact-loading, syringe-loading, and hypertonic loading and hypotonic loading. Mechanical methods such as loading). Additionally, cells can be genetically engineered to express reporter molecules, such as GFP, bound to the protein of interest, as described above (Chalfie and Prasher US Pat. No. 5,491,084; Cubitt et al. (1995), Trends in Biochemical Science 20: 448-455.

In addition, certain cell types within an individual may include components that can be specifically labeled with the preparation of stilbarts or analogs thereof that may not occur in other cell types. For example, epithelial cells often contain polarized membrane components. That is, these cells asymmetrically distribute macromolecules along their plasma membrane. Connective or support tissue cells often include granules that are trapped with molecules specific to that cell type (eg, heparin, histamine, serotonin, etc.). Most muscle tissue cells contain sarcoplasmic reticulum, a specialized organelle that functions to regulate the concentration of calcium ions in the cytoplasm. Many neural tissue cells include secretory granules and vesicles entrapped with neurohormones or neurotransmitters. Thus, fluorescent molecules can be designed to label specific cells within a population of mixed cell types, as well as specific components within specific cells.

Those skilled in the art will recognize various methods of measuring fluorescence. For example, some fluorescence reporter molecules show excitation or emission spectra changes, some fluorescence of one fluorescence reporter, and a resonance energy transfer with increased fluorescence of the second fluorescence reporter. , Some show loss (quenching) or expression of fluorescence, and some report rotational movement (Giuliano et al. (1995), Ann. Rev. of Biophysics and Biomol. Structure 24: 405-434; Giuliano et al. (1995), Methods in Neuroscience 27: 1-16).

In biochemical, molecular biology, and medical diagnostics, it is often desirable to add fluorescent labels to proteins so that the proteins can be easily tracked and quantified. Conventional methods of labeling require the protein to be reacted covalently with fluorescent dyes in vitro and to be reconditioned to remove excess dye and damaged proteins. When labeled proteins are used in cells, the proteins are typically microinjeciton; This can be a difficult and / or time-consuming task that cannot be practically performed for many cells.

The compounds of the present invention can be used as staining agents for isolated DNA / RNA or for isolated cells and organelles. In addition, the compounds of the present invention can be used as nuclear staining agents in cell based assays. Still other applications include using the compounds of the present invention to stain tissues and / or organs in embryos, larvae, nematodes, insects and other parasites and whole animals. In some embodiments, the compounds of the present invention may complex with specific antibodies for imaging of specific organ tissues in an entire animal model system. As used herein in various grammatical forms, the term “antibody” or “antibody molecule” refers to immunoglobulin molecules (including IgG, IgE, IgA, IgM, IgD) and / or immunologically active portions of immunoglobulin molecules That is, it refers to a molecule containing an antibody binding site or paratope and capable of binding an antigen. An "antibody combining site" or "antigen binding site" refers to an antibody molecule consisting of the variable and hypervariable regions (CDRs) of the heavy and light chains that specifically bind antigens. Structural part. As is known to those skilled in the art to which this invention pertains, certain properties of antibodies relate to immunoglobulin isotypes. In an exemplary embodiment, the antibody or antigen-binding fragment is an IgG2a, IgGl or IgG2b isotype molecule. Antibodies or fragments thereof may also be used in birds (eg, chickens, turkeys, ducks, geese, quails, etc.) and mammals (eg, humans, non-human primates, mice, mice, earthenware, cattle, goats, sheep, Horses, pigs, dogs, cats, etc.) species.

The present invention combines multiple cell screening formats with fluorescence-based molecular reagents and computer-based feature extraction, data analysis, and automation to increase the amount and speed of data collection, reduce cycle times, and ultimately It is possible to provide systems, methods and screening that combine high efficiency screening (HTS) and high content screening (HCS) to improve target validation and candidate optimization, resulting in faster assessments.

Thus, in some embodiments, the invention provides a cell and, thus, a nucleus, vesicular antifoam and / or rough endoplasmic reticulum, centrosome, cytoskeleton, cell wall, cell membrane, flagella, cilia, chloroplast, mitochondria, Golgi apparatus, ribosomes, lysosomes, centrioles , Acrosome, glyoxysome, secretory vesicles, peroxysomes, vacuoles, melanosomes, myofibers and parenthesomes. In another embodiment, the invention features techniques for determining the presence, location, amount, and / or health of analytes and microorganisms.

In another embodiment, the invention features a method of determining the presence, position, or amount of a desired RNA molecule in a cell or in vitro sample. The method comprises the steps of: (a) expressing a fusion RNA molecule comprising the target RNA molecule covalently linked to an RNA aptamer in a cell or sample, (b) the RNA aptamer binds to a fluorophore and Contacting the cell or sample with a fluorophore under conditions such that fluorescence can be increased or decreased thereby, and (c) visualizing fluorescence of the fluorophore.

In another embodiment, the invention features a method of determining the presence, position, or amount of a target DNA molecule in a cell or in vitro sample. The method comprises the steps of: (a) expressing a fusion DNA molecule comprising the target DNA molecule covalently linked to a DNA aptamer in a cell or sample, (b) the DNA aptamer binds to and is fluorescence by fluorophores Contacting the cell or sample with a fluorophore under conditions that allow for the increase or decrease thereof, and (c) visualizing the fluorescence of the fluorophore.

Embodiments of the invention are also assays for determining the presence or absence of cells, analytes, nucleic acids or microorganisms in a sample, such as a fluid or aqueous sample suspected of containing a microorganism, wherein the assay is stained. In order to provide a stained sample comprising the cells, analytes, nucleic acids or microorganisms, the dyes directly dye the cells, analytes, nucleic acids or microorganisms, the dye is a compound represented by the following formula I,

In formula I, the NR ₁ R ₂ and NR ₃ R ₄ moieties are in the ortho, meta or para position; X ⁻ is an anionic salt; X ⁻ is fluoride, chloride, bromide, iodide halide, mesylate, tosylate, naphthylate, nosylate, para-aminobenzoate, benzenesulfonate, besylate, lauryl sulfate, 2,4- Selected from the group consisting of dihydroxy benzophenone, 2- (2-hydroxy-5'-methylphenyl) benzotriazole, ethyl 2-cyano-3,3-diphenyl acrylate and 5-butyl phenyl salicylate Become; R _1, R _2, R _3, or R ₄ is methyl, ethyl, C _{1 -10} alkyl (straight or branched), alkenes (linear or branched), or a member selected from the group consisting of, or taken with R ₁ And R ₂ or R ₃ and R ₄ together with the nitrogen atom to which they are attached form a pyrrolidino or piperidino ring; R ₅ is a polyalkylene glycol moiety, C _{1 -10} alkyl (straight or branched), alkenes (linear or branched), alkynes, substituted aryl moiety and unsubstituted aryl moieties, substituted benzyl Moieties and unsubstituted benzyl moieties and / or organometallic moieties, or R ₅ may also be an organometallic compound such as organotin, organosilicon, or organo magnesium, and in addition, R ₅ is n to 1 to Is a number of 6, M is an organometallic compound such as tin, silicon or germanium, and R ₆ can be (CH ₂ ) _n -MR ₆ selected from propyl, butyl, or any alkyl compound Combining the sample with the reagent for forming a labeled cell, nucleic acid or microorganism; Contacting the dyed sample; And observing accumulation of the stained cells, analytes, nucleic acids or microorganisms. The dye may be encapsulated. In some embodiments, the dye is loaded into lipid vesicles such as microcapsules or liposomes, micelles and / or vissels, respectively, to form microcapsule formulations or lipid vesicle formulations. The dye may also be PEGylated.

In various embodiments of the invention, the cells can be bound by one of the compounds according to formula I. In some embodiments, the cell is a prokaryotic cell, such as a Gram-negative bacterial cell or a Gram-positive bacterial cell. In other embodiments, the cell is a eukaryotic cell. For example, the cells may be yeast, Caenorhabditis, Frog (Xenopus), Drosophila, zebrafish, squid, plant, mammalian cell, embryonic cell or human cell. In another embodiment, the cell or sample is contacted with the fluorophore by incubating the cell or sample with the fluorophore. In another embodiment, fluorophores are injected into cells or administered to plants, embryos, mammals, transgenic animals, or humans comprising the cells.

In other embodiments, the population of nucleic acids comprises more than one DNA molecule or more than one RNA molecule. Nucleic acids can have natural or non-natural polynucleotide sequences. Regions of nucleic acids, such as loops, tetraloops, or helixes, or portions thereof, comprise random sequences that differ between some or all members in a population. In other embodiments, the sequence of the loop, tetatarup, or helix is the same for all members of the population. The length of any loop or helix may be the same or different between members of the population. The population of nucleic acids may comprise any number of unique molecules. For example, the population may include about 10, 10 ² , 10 ⁹ , or 10 ¹¹ unique molecules or about 10 ¹³ , 10 ¹⁵ , 10 ²⁰ , or more unique molecules. In some embodiments, one or more polynucleotide sequences are non-natural sequences. The nucleic acids may all have the same length or some molecules may have different lengths.

"Fluorophore" means a compound capable of emitting a fluorescent signal. As described herein, fluorophores used in the present invention have a higher fluorescence intensity when bound to a nucleic acid or protein than when present in solution unbound. The fluorescence intensity of the bound fluorophores may be about 1, 5, 10, 50, 100, 500, or 1000 times the fluorescence intensity of the free fluorophores in the aqueous solution. Examples of conditions that can enhance fluorescence of bound fluorophores include rigidification, conformational restriction, and sequestration from solution. In one embodiment, the fluorophores do not bind covalently to the aptamers.

Other fluorophores have lower fluorescence when bound to nucleic acids or proteins than when free in solution. The fluorescence intensity of the bound fluorophores may be about 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, or 1000 times lower than the fluorescence intensity of the free fluorophores in the aqueous solution. An example of a condition that can reduce the fluorescence of a bound fluorophore is a structural change in the fluorophore that reduces the fluorescence intensity of the fluorophore. In one embodiment, the fluorophores do not bind covalently to the aptamers.

Other fluorophores used in multiplexing, such as calcium-sensing dyes, can take on two or more different structural states resulting in different fluorescence intensities. The aptamers of the present invention can control the fluorescence of fluorophores by increasing the percentage of fluorophores in a particular structural state with increased or decreased fluorescence.

Phosphorophores can be dissolved in aqueous solutions at concentrations of about 0.1 μM, 1 μM, 10 μM and 50 μM. Incubation of cells with such concentrations of fluorophores does not affect the viability of the cells. In another embodiment, incubating the cells with such a fluorophore for about 1 or 2 hours to about 8, 12, 24, 36 hours or more to inactivate, replicate, transcription or translate intracellular proteins. It does not require the presence of another compound to prevent the toxic effects of fluorophores such as inhibition of, or induction of cell death. "Cell permeable" means that it can migrate through the cell membrane or cell wall into the cytoplasm or periplasm of the cell by active or passive diffusion. Fluorophores can migrate through both the outer and inner membranes of Gram-negative bacteria or through the cell walls and plasma membranes of plant cells. In addition, fluorophores can be used to visualize cells, analytes and / or nucleic acids in a sample in vitro.

Embodiments of the invention are nucleic acid probes comprising a single-stranded oligonucleotide and a compound represented by formula I

In formula I, the NR ₁ R ₂ and NR ₃ R ₄ moieties are in the ortho, meta or para position;

X ⁻ is an anionic salt;

R ₁ , R ₂ , R ₃ , or R ₄ are independently selected from the group consisting of methyl, ethyl, C _1-10 alkyl (linear or branched), alkene (linear or branched), or taken together R ₁ and R ₂ or R ₃ and R ₄ together with the nitrogen atom to which they are attached form a pyrrolidino or piperidino ring;

R ₅ is a polyalkylene glycol moiety, C _1-10 alkyl (linear or branched), alkene (linear or branched), alkyne, substituted aryl moiety and unsubstituted aryl moiety, substituted benzyl Moieties and unsubstituted benzyl moieties and / or organometallic moieties, or R ₅ may also be an organometallic compound such as organotin, organosilicon, or organo magnesium, and in addition, R ₅ is n to 1 to Is a number of 6, M is an organometallic compound such as tin, silicon or germanium, and R ₆ is a nucleic acid that may be (CH ₂ ) _n -MR ₆ selected from propyl, butyl, or any alkyl compound It may comprise a probe.

Single-stranded oligonucleotides may be DNA oligomers. The phosphorus atoms of the DNA oligomers can be linked by chemical bonds via linkers. Single-stranded oligonucleotides may have sequences that are complementary to a particular sequence in a target nucleic acid comprising the particular sequence.

An embodiment of the invention is a method of selecting a nucleic acid molecule that binds to a compound of formula I, wherein the binding increases the fluorescence intensity of the compound of formula I, wherein the method comprises:

(a) providing a population of candidate nucleic acid molecules;

(b) selecting the candidate nucleic acid molecule that binds to the compound of Formula I;

(c) contacting the compound of formula I with the candidate nucleic acid molecule that binds to the compound of formula I; And

(d) selecting said nucleic acid molecule which, upon binding to the compound of formula I, increases its fluorescence intensity.

The nucleic acid can be DNA or RNA.

Another embodiment of the invention is a method of determining the presence, position, or amount of a target nucleic acid in a cell or in vitro sample, the method comprising: (a) expressing the target nucleic acid in the cell or the sample; (b) contacting the cell or sample with a compound of formula I, thereby binding the compound to the compound of formula I and increasing its fluorescence intensity; And (c) visualizing or measuring the fluorescence of the compound of formula I, thereby determining the presence, location or amount of the desired nucleic acid in the cell or in vitro sample.

An embodiment of the invention is a method of determining whether a compound of formula I can modulate transcription of a target nucleic acid, the method comprising: (a) expressing a target nucleic acid in the cell or the sample; (b) contacting the cell or sample with a compound of formula I, thereby binding the compound to the compound of formula I and increasing its fluorescence intensity; And (c) determining the fluorescence intensity in the presence and absence of the compound, whereby it is determined to control the transcription when the compound causes a change in the fluorescence intensity. It is a method that is.

Another embodiment is a kit for staining nucleic acids or amino acids in a sample, comprising: (a) a dyeing mixture comprising one or more dyes forming a combined mixture, wherein each dye independently has formula I; (b) instructions for combining said dye or dyes with a sample comprising or appearing to contain a nucleic acid or amino acid, said instructions comprising: i) containing said dye or dyes in a sample that appears to contain a nucleic acid or amino acid. Combining with a dyeing mixture to form a combined mixture; And ii) incubating the combined mixture for a time sufficient to cause the dye in the dyeing mixture to bind a nucleic acid or amino acid in the sample mixture to produce a dye-amino acid or dye-nucleic acid complex that provides a detectable optical response upon irradiation. It includes a kit comprising instructions that comprises the step of making.

A further embodiment is a method of detecting a target analyte in a sample comprising or suspected of containing one or more analytes, comprising: (a) providing a sample on a solid support wherein the analyte is a nucleic acid molecule; (b) combining the sample with a specific-binding molecule, wherein (i) the specific-binding molecule is a polymerase chain reaction amplification product comprising biotin as a detectable label, and (ii) the combination is When present with a specific-binding molecule, is performed under conditions that allow the formation of a first complex comprising said analyte; (c) removing unbound specific-binding molecules; (d) providing a compound having Formula I; And (e) detecting an optical response based on the binding of said compound.

Embodiments of the invention include providing an array of positions comprising a plurality of cells comprising one or more fluorescent reporter molecules; Scanning a plurality of cells at each of the positions comprising the cell to obtain a fluorescence signal from a fluorescent reporter molecule within the cell; Converting the fluorescence signal into digital data; And determining the distribution, environment, or activity of fluorescent reporter molecules in the inclusions using the digital data.

Embodiments of the invention can also be used in cell arrays. Cell arrays can be used to screen multiple compounds with activity for specific biological functions, including preparing arrays of cells for parallel handling of cells and reagents. A standard 96 well microtiter plate, 86 mm x 129 mm with a 6 mm diameter well on a 9 nm pitch, is used for current automated loading and robotic handling systems. The microplates are typically 20 mm x 30 mm and the cell location has a diameter of 100-200 microns on a distance of about 500 microns. Methods of making microplates are disclosed in US Pat. No. 6,103,479. The microplates may consist of a coplanar layer of material to which cells attach, or may consist of an etched three-dimensional surface of similarly patterned material. The terms "well" and "microwell" refer to locations within an array of structures in which cells attach and within which cells are imaged. The microplates may also include fluid delivery channels in the spaces between the wells. The smaller format of the microplates increases the overall efficiency of the system by minimizing the overall movement required for the amount of reagents, storage and handling and scanning operations during manufacture. In addition, the entire area of the microplate can be imaged more efficiently, allowing a second mode of operation for the microplate reader as described below herein.

As described above, according to the present invention, when used in the detection of nucleic acids, upon intercalation into double-stranded nucleic acids, there can be a large increase in fluorescence and a large difference between excitation and emission wavelengths (ie, It is possible to provide novel compounds which are novel fluorescent dyes with Stokes shift. The compounds are used for conventional nucleic acid assays by contacting with double-stranded nucleic acids or by linking them to single-stranded oligonucleotides to form nucleic acid probes.

The compounds of the present invention may be characterized in that their fluorescence spectra do not overlap with the fluorescence spectra of known fluorescent intercalative dyes. Thus, the combined use of two or more nucleic acid probes using a compound of the invention and commonly known fluorescent insert dye (s) allows amplification of two or more target nucleic acids in a sample in a closed container without separation while amplifying the target nucleic acid. It is possible to measure the amplification product from, ie, to measure two or more target nucleic acids simultaneously.

The invention is explained in more detail in the following examples. This embodiment is intended as an illustration of the invention and should not be understood as limiting the invention.

1A-1L show confocal images of murine mammary carcinoma 4T1 cells incubated with a compound according to some embodiments of the invention (FIGS. 1B, 1F and 1J) and Hoechst for staining nuclei. The images stained together (FIGS. 1 a, 1 e and 1 i, blue), the images stained with MitoTracker Deep Red to stain mitochondria (FIGS. 1 c, 1 g and 1 k, shown green in the image) are shown. FIG. 1D shows an overlaid image of the image shown in FIGS. 1A-1C. FIG. 1H shows a superimposed image of the image shown in FIGS. 1E-1G. FIG. 1L shows a superimposed image of the image shown in FIGS. 1I-1K.

Spectra were obtained for a solution at room temperature (22 ° C.) in a methacrylate cuvette with 1 cm path length in a Beckman DU-640 spectrometer (Beckman-Coulter, Palo Alto CA). The scan rate was 10 nm / min from 800 nm to 250 nm. A blank scan was performed with the same diluent used for the analyte and subtracted it from the spectrum of the compound.

A liposome formulation (a turbid colored solution) was provided comprising stilvax chloride (red powder) and stilvax. Ethidium bromide, DMSO and ethanol were purchased from Sigma-Aldrich (St. Louis, Mo). Methacrylate 1.5 mL cuvette was purchased from Fisher Scientific. Molar absorption (ε) was calculated by dividing the observed optical density by the number of moles of analyte ([molarity] * volume), which represents a theoretical absorbance of 1.0 mole of test material.

When dissolved in absolute ethanol, stilbart chloride produced a dark red solution. A second dilution with anhydrous ethanol or 70% (aq.) Ethanol was performed before performing the analysis. In both cases, one broad absorption peak with an apparent maximum of 509-511 nm and one broad peak with a molar extinction coefficient of ε = 6638400 were observed.

The molecules of the present invention were similarly soluble in DMSO to form a solution with dark red at an initial concentration of 4.1 mg / mL. A spectrum showing one broad peak of ε = 4401574 at 507 nm was obtained after secondary dilution with the same solvent up to a final concentration of 50 μM.

Stilvax chloride was found to be almost insoluble in water. Thus, spectral measurements were performed in ethanol and DMSO solutions. Of these two solvents, greater absorption was observed when dissolved in ethanol solution than the compound dissolved in DMSO (ε = 6638400 Vs. 4401574). Secondary dilution with 70% aqueous ethanol solution was the same as the result of dilution with anhydrous ethanol.

Liposomal formulations provided as yellow to pink solutions were added directly to a cuvette with water (50 μl in 1.0 mL) to produce an apparent light scattering turbidity. The resulting spectrum had a maximum at 585 nm with ε = 302400. When formulated into a water soluble liposome complex, the formulation became slightly cloudy when added to water at a dilution of 1:20. The resulting spectrum showed a shift in wavelength and had a maximum optical density and ε = 302400 at 585 nm. Reduced molar absorbance is not surprising because the analyte is contained in the liposome suspension. Light scattering at the surface of liposomes will interfere with the compound.

For comparison, ethidium bromide, a DNA binding dye dissolved in water, had a maximum optical density with epsilon = 6602400 at 284 nm and a much smaller peak with epsilon = 700460 at 478 nm.

The compounds of the present invention can fluoresce in the range of about 400 nm to 700 nm.

Cell staining

The Ukrainian breast carcinoma 4T1 cell population was individually incubated with the three compounds according to the invention for about 30 minutes, followed by co-staining with HOECHST (blue) and MitoTracker Deep Red (green). Confocal images were taken with Zeiss Meta 510 LSM. The dyes were excited by a laser at a wavelength of 540 nm (red).

1A-1L show confocal images of murine mammary carcinoma 4T1 cells incubated with a compound according to some embodiments of the invention (FIGS. 1B, 1F and 1J) and Hoechst for staining nuclei. Images stained together (FIGS. 1A, 1E and 1I, Blu), images stained with MitoTracker Deep Red to stain mitochondria (FIGS. 1C, 1G and 1K, shown green in the image) are shown. FIG. 1D shows an overlaid image of the image shown in FIGS. 1A-1C. FIG. 1H shows a superimposed image of the image shown in FIGS. 1E-1G. FIG. 1L shows a superimposed image of the image shown in FIGS. 1I-1K. Overlapping images indicate that the compounds of the present invention can stain cells, in particular mitochondria, in either a live cell population or a fixed cell population.

The foregoing is illustrative of the invention and is not to be construed as limiting the invention. While exemplary embodiments of the invention have been described, those skilled in the art will clearly understand that many modifications are possible in the above exemplary embodiments without departing from the novel teachings and advantages of the invention. Accordingly, all such modifications are intended to be included within the scope of this invention as defined in the claims. The invention is defined by the following claims, with equivalent scope of the claims to be included therein.

Claims (25)

An assay for determining the presence, absence or health of cells, analytes, nucleic acids or microorganisms in a sample, the assay comprising A dye that stains said cell, analyte, nucleic acid, or microorganism to provide a stained sample comprising the stained cell, analyte, nucleic acid, or microorganism, said dye being a compound represented by Formula I below :

In formula I, the NR ₁ R ₂ and NR ₃ R ₄ moieties are in the ortho, meta or para position; X ⁻ is an anionic salt; _{R 1, R 2, R 3} , or R ₄ are independently selected from the group consisting of methyl, ethyl, C _{1 -10} alkyl (straight or branched), alkenes (linear or branched), or taken together R ₁ and R ₂ or R ₃ and R ₄ together with the nitrogen atom to which they are attached form a pyrrolidino or piperidino ring; R ₅ is methyl, ethyl, C _{1 -10} alkyl (straight or branched), alkenes (linear or branched), alkynes, n- propyl, i- propyl, n- butyl, i- butyl, an organometallic compound And a labeling reagent selected from the group consisting of polyalkylene glycol moieties, substituted aryl moieties and unsubstituted aryl moieties, and substituted benzyl moieties and unsubstituted benzyl moieties. Combining the samples to form labeled cells, nucleic acids or microorganisms; And Observing the accumulation of said stained cells, analytes, nucleic acids or microorganisms. The assay of claim 1, wherein the dye is encapsulated or pegylated. The method of claim 1, wherein R ₅ is n is a number of 1 to 6, M is an organometallic compound selected from the group consisting of tin, silicon and germanium, R ₆ is substituted or unsubstituted propyl, substituted or unsubstituted the butyl, and in the (CH2) _n -MR ₆ would be substituted or unsubstituted selected from the group consisting of substituted alkyl Method. The assay of claim 1 wherein the compound of formula I has the structure

The assay of claim 1 wherein the compound of formula I has the structure

The assay of claim 1 wherein the compound of formula I has the structure

As a probe comprising a ligand or an antibody and a compound of formula I:

In formula I, the NR ₁ R ₂ and NR ₃ R ₄ moieties are in the ortho, meta or para position; X ⁻ is an anionic salt; R ₁ , R ₂ , R ₃ , or R ₄ are independently selected from the group consisting of methyl, ethyl, C _1-10 alkyl (linear or branched), alkene (linear or branched), or taken together R ₁ and R ₂ or R ₃ and R ₄ together with the nitrogen atom to which they are attached form a pyrrolidino or piperidino ring; R ₅ is methyl, ethyl, C _{1 -10} alkyl (straight or branched), alkenes (linear or branched), alkynes, n- propyl, i- propyl, n- butyl, i- butyl, an organometallic compound , Polyalkylene glycol moiety, substituted aryl moiety and unsubstituted aryl moiety, and substituted benzyl moiety and unsubstituted benzyl moiety; And Wherein said compound is bound to said ligand or antibody by chemical bonding. The probe of claim 7 wherein the ligand is an oligonucleotide. The probe of claim 8, wherein the oligonucleotide is a DNA oligomer. The probe of claim 9 wherein the phosphorus atom in the DNA oligomer is bound to the dye. The probe of claim 8, wherein the oligonucleotide has a sequence complementary to the particular sequence of the target nucleic acid comprising the specific sequence. As a method of selecting an analyte that binds to a compound of Formula I:

In formula I, the NR ₁ R ₂ and NR ₃ R ₄ moieties are in the ortho, meta or para position; X ⁻ is an anionic salt; R ₁ , R ₂ , R ₃ , or R ₄ are independently selected from the group consisting of methyl, ethyl, C _1-10 alkyl (straight or branched), alkene (straight or branched), or taken together R ₁ and R ₂ or R ₃ and R ₄ together with the nitrogen atom to which they are attached form a pyrrolidino or piperidino ring; R ₅ is methyl, ethyl, C _{1 -10} alkyl (straight or branched), alkenes (linear or branched), alkynes, n- propyl, i- propyl, n- butyl, i- butyl, an organometallic compound , Polyalkylene glycol moiety, substituted aryl moiety and unsubstituted aryl moiety, and substituted benzyl moiety and unsubstituted benzyl moiety; Said binding increases the fluorescence intensity of the compound of formula I and the method comprises: (a) providing a population of analytes; (b) selecting the analyte that binds to the compound of Formula I; (c) contacting the analyte that binds to the compound of Formula I; And (d) selecting the analyte that increases the fluorescence intensity of the analyte upon binding to the compound of Formula I. The method of claim 12, wherein the analyte is DNA. The method of claim 12, wherein the analyte is RNA. The method of claim 12, wherein the analyte is a small molecule. The method of claim 12, wherein the analyte is a microorganism. The method of claim 12, wherein the analyte is a cell. A method of determining whether a nucleic acid of interest interacts with a protein of interest in a cell or in vitro sample, the method comprising: (a) has the following formula I,

In formula I, the NR ₁ R ₂ and NR ₃ R ₄ moieties are in the ortho, meta or para position; X ⁻ is an anionic salt; _{R 1, R 2, R 3} , or R ₄ are independently selected from the group consisting of methyl, ethyl, C _{1 -10} alkyl (straight or branched), alkenes (linear or branched), or taken together R ₁ and R ₂ or R ₃ and R ₄ together with the nitrogen atom to which they are attached form a pyrrolidino or piperidino ring; R ₅ is methyl, ethyl, C _{1 -10} alkyl (straight or branched), alkenes (linear or branched), alkynes, n- propyl, i- propyl, n- butyl, i- butyl, an organometallic compound Nucleic acid app that binds to a first compound selected from the group consisting of: polyalkylene glycol moieties, substituted aryl moieties and unsubstituted aryl moieties, and substituted benzyl moieties and unsubstituted benzyl moieties. Expressing in said cell or sample a fusion nucleic acid comprising said target nucleic acid covalently linked to a tamper; (b) expressing in said cell or said sample a fusion protein comprising said target protein covalently linked to a detectable protein that binds to said second compound having Formula I, said first compound having Formula I The emission wavelength of the compound is different from the emission wavelength of the second compound having Formula I, and the emission wavelength of the first compound having Formula I induces fluorescence of the second compound having Formula I or The emission wavelength of the second compound having formula I induces fluorescence of the first compound having formula I; (c) the cell or the sample, i) alone with a first compound having formula I and a second compound having formula I, or ii) with a first compound having formula I, or iii Contacting with a second compound having Formula I alone, thereby binding the nucleic acid aptamer to the first compound having Formula I and increasing its fluorescence intensity, whereby the detectable protein is Binding to a second compound having I and increasing its fluorescence intensity; And (d) measuring the fluorescence intensity of the first compound having formula I in the presence and absence of a second compound having formula I or in the presence and absence of the first compound having formula I When the fluorescence intensity of the second compound having I is measured, whereby fluorescence resonance energy transfer occurs between the first compound having Formula I and the second compound having Formula I, the target nucleic acid may be And determining which one interacts with the protein. The method of claim 18, further comprising the following steps: (a) expressing a fusion protein comprising said target protein covalently linked to a detectable protein having intrinsic fluorescence in said cell or said sample, wherein the emission wavelength of said compound of formula I is determined by Wherein the emission wavelength of the compound of formula I induces fluorescence of the detectable protein or the emission wavelength of the detectable protein induces fluorescence of the compound of Formula I; (b) contacting said cell or said sample with said compound of formula I, whereby said nucleic acid aptamer binds to said compound of formula I and increases its fluorescence intensity; And (c) measuring the fluorescence intensity of the compound of formula I in the presence and absence of the detectable protein or measuring the fluorescence intensity of the detectable protein in the presence and absence of the compound of formula I, whereby When fluorescence resonance energy transfer occurs between the compound of formula I and the detectable protein, the target nucleic acid is determined to interact with the target protein. A method of determining the presence or absence of one or more target compounds in a sample, wherein the compound is a fluorescent molecule of a compound of Formula I:

In formula I, the NR ₁ R ₂ and NR ₃ R ₄ moieties are in the ortho, meta or para position; X ⁻ is an anionic salt; R ₁ , R ₂ , R ₃ , or R ₄ are independently selected from the group consisting of methyl, ethyl, C _1-10 alkyl (linear or branched), alkene (linear or branched), or taken together R ₁ and R ₂ or R ₃ and R ₄ together with the nitrogen atom to which they are attached form a pyrrolidino or piperidino ring; R ₅ is methyl, ethyl, C _{1 -10} alkyl (straight or branched), alkenes (linear or branched), alkynes, n- propyl, i- propyl, n- butyl, i- butyl, an organometallic compound , Polyalkylene glycol moiety, substituted aryl moiety and unsubstituted aryl moiety, and substituted benzyl moiety and unsubstituted benzyl moiety; The method is: Providing a plurality of electrophoretic probes specific for said one or more target compounds, each having a target-binding moiety; Combining the sample with the plurality of electrophoretic probes in the presence of a target compound such that a complex is formed between each target compound and one or more electrophoretic probes specific thereto; And Isolating and identifying said compound to determine the presence or absence of said one or more target compounds. A kit for staining cells, analytes, nucleic acids or microorganisms in a sample, (a) at least one dye to form a combined mixture, wherein the at least one dye has the formula

In formula I, the NR ₁ R ₂ and NR ₃ R ₄ moieties are in the ortho, meta or para position; X ⁻ is an anionic salt; R ₁ , R ₂ , R ₃ , or R ₄ are independently selected from the group consisting of methyl, ethyl, C _1-10 alkyl (straight or branched), alkene (straight or branched), or taken together R ₁ and R ₂ or R ₃ and R ₄ together with the nitrogen atom to which they are attached form a pyrrolidino or piperidino ring; R ₅ is methyl, ethyl, C _{1 -10} alkyl (straight or branched), alkenes (linear or branched), alkynes, n- propyl, i- propyl, n- butyl, i- butyl, an organometallic compound Dyeing mixtures selected from the group consisting of polyalkylene glycol moieties, substituted aryl moieties and unsubstituted aryl moieties, and substituted benzyl moieties and unsubstituted benzyl moieties; And b) i) combining a sample of cells, analytes, nucleic acids and / or microorganisms with a dyeing mixture comprising said at least one dye to form a combined mixture; And   ii) the combined mixture is allowed to react for a period of time sufficient for the dye in the dye mixture to bind to cells, analytes, nucleic acids or microorganisms in the sample mixture, resulting in a stained cell that produces a detectable optical response upon irradiation And instructions for combining said dye or dyes with a sample comprising cells, analytes, nucleic acids and / or microorganisms, the method comprising forming water, nucleic acids or microorganisms. The kit of claim 21, wherein the kit is used for cell differentiation. As a compound of Formula I:

In formula I, the NR ₁ R ₂ and NR ₃ R ₄ moieties are in the ortho, meta or para position; X ⁻ is an anionic salt; _{R 1, R 2, R 3} , or R ₄ are independently selected from the group consisting of methyl, ethyl, C _{1 -10} alkyl (straight or branched), alkenes (linear or branched), or taken together R ₁ and R ₂ or R ₃ and R ₄ together with the nitrogen atom to which they are attached form a pyrrolidino or piperidino ring; R ₅ is methyl, ethyl, C _{1 -10} alkyl (straight or branched), alkenes (linear or branched), alkynes, n- propyl, i- propyl, n- butyl, i- butyl, an organometallic compound , Polyalkylene glycol moiety, substituted aryl moiety and unsubstituted aryl moiety, and substituted benzyl moiety and unsubstituted benzyl moiety; And Said compound is encapsulated or pegylated. The compound of claim 23, wherein the encapsulated or pegylated compound is water soluble. The compound of claim 23, wherein the compound fluoresces in the range of about 400 nm to 700 nm.

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